Application
Western blot
Loading amount
20 µg
Gel Running Conditions
Reduced Denaturing (8% acrylamide)
Sample
Pig Tissue lysate - other (Muscle sarcoplasmic extract)
Specification
Muscle sarcoplasmic extract
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C
Other product details
Incubation time
15 hour(s) and 0 minute(s) · Temperature: 4°C · Diluent: PBS-Tween
Dilution
1/40000
Secondary antibody
Name
Non-Abcam antibody was used: Goat Anti-Mouse IgG (Fc specific)–Peroxidase antib
Host species: Goat
Clonality: Polyclonal
Conjugation: Horse Radish Peroxidase
Host species: Goat
Clonality: Polyclonal
Conjugation: Horse Radish Peroxidase
Dilution
1/10000
Detection
Detection method
ECL Prime
Bands
Specific: 70 kDa
Exposure
2 minute(s) and 0 second(s)
Additional data
Additional Notes
This antibody is supposed to detect Hsp70 as well as Grp75 and Hsc70. In our normal gel runs we could only resolve this as one band. However, with some work, using less protein, less antibody, a lower acrylamide percentage, and larger gels with longer run times, we have been able to detect two bands, which we assume are Grp75 and Hsp70.
In part A of the attached image is the antibody after protein was run and transferred from a 12.5% acrylamide gel with titrations of 1:10,000 (Lanes 1 and 2), 1:20,000 (Lanes 4 and 5), and a secondary control (no primary antibody, Lanes 7 and 8). 40 ug protein was loaded in each lane. Part B shows our results with better resolution. Hsp70 antibody is diluted to 1:40,000, protein load is decreased to 20 ug, acrylamide is reduced to 8%, and the gel size/run time is increased from approximately 300 Vh to approximately 1200 Vh.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.
Dr. Shannon Cruzen
Verified customer
投稿 Mar 27 2014